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III
Microbial
Metabolism
Chapter 8
Metabolism: Energy, Enzymes, and
Regulation
CHAPTER
8
Metabolism:
Energy, Enzymes,
and Regulation
Chapter 9
Metabolism: Energy Release and
Conservation
Chapter 10
Metabolism:The Use of Energy in
Biosynthesis
This diagram shows
E. coli aspartate
carbamoyltransferase
in the less active
T state. The catalytic
polypeptide chains are
in blue and the
regulatory chains are
colored red.
Outline
8.1 Energy and Work 154
8.2 The Laws of
Thermodynamics 155
8.3 Free Energy and
Reactions 156
8.4 The Role of ATP in
Metabolism 157
8.5 Oxidation-Reduction
Reactions and Electron
Carriers 157
8.6 Enzymes 161
Structure and Classification
of Enzymes 161
The Mechanism of Enzyme
Reactions 161
The Effect of Environment
on Enzyme Activity 162
Enzyme Inhibition 164
8.7 The Nature and Significance
of Metabolic Regulation 164
8.8 Metabolic Channeling 165
8.9 Control of Enzyme
Activity 165
Allosteric Regulation 165
Covalent Modification of
Enzymes 167
Feedback Inhibition 169
Concepts
1. Energy is the capacity to do work. Living
organisms can perform three major types of
work: chemical work, transport work, and
mechanical work.
2. Most energy used by living organisms originally
comes from sunlight trapped during
photosynthesis by photoautotrophs.
Chemoheterotrophs then consume autotrophic
organic materials and use them as sources of
energy and as building blocks.
3. An energy currency is needed to connect energyyielding exergonic reactions with energyrequiring endergonic reactions. The most
commonly used currency is ATP.
4. All living systems obey the laws of
thermodynamics.
5. When electrons are transferred from a reductant
with a more negative reduction potential to an
oxidant with a more positive potential, energy is
made available. A reversal of the direction of
electron transfer—for example, during
photosynthesis—requires energy input.
6. Enzymes are protein catalysts that make life
possible by increasing the rate of reactions at
ambient temperatures. Enzymes do not change
chemical equilibria or violate the laws of
thermodynamics but accelerate reactions by
lowering their activation energy.
154
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
7. Metabolism is regulated in such a way that (a) cell components are
maintained at the proper concentrations, even in the face of a changing
environment, and (b) energy and material are conserved.
8. The localization of enzymes and metabolites in separate compartments of a
cell regulates and coordinates metabolic activity.
9. The activity of regulatory enzymes may be changed through reversible
binding of effectors to a regulatory site separate from the catalytic site or
through covalent modification of the enzyme. Regulation of enzyme
activity operates rapidly and serves as a fine-tuning mechanism to adjust
metabolism from moment to moment.
10. A pathway’s activity is often controlled by its end products through
feedback inhibition of regulatory enzymes located at the start of the
sequence and at branch points.
Light energy
Chemical energy
Photolithoautotrophs
Chemolithoautotrophs
CO2
Organic
compounds
Heterotrophs and Autotrophs
Living cells are self-regulating chemical engines, tuned to operate on
the principle of maximum economy.
—A. L. Lehninger
hapters 3 and 4 contain many examples of an important principle: that a cell’s structure is intimately related to its function. In each instance one can readily relate an organelle’s
construction to its function (and vice versa). A second unifying principle in biology is that life is sustained by the trapping and use of energy, a process made possible by the action of enzymes. Because this
is so crucial to our understanding of microbial function, considerable
attention is given to energy and enzymes in this chapter.
The organization of microbial metabolism will be briefly described in chapters 8 to 10. Metabolic pathways are treated as a
sequence of enzymes functioning as a unit, with each enzyme using as its substrate a product of the preceding enzyme-catalyzed
reaction. This picture of metabolic pathways is incomplete because we will usually ignore the regulation of pathway operation
for the sake of space and simplicity. However, one should keep in
mind that both regulation of the activity of individual pathways
and coordination of the action of separate sequences are essential
to the existence of life. Cells become disorganized and die without adequate control of metabolism, and regulation is just as important to life as is the efficient use of energy. Thus the last part
of this chapter will be devoted to the regulation of metabolism as
a foundation for the subsequent discussion of pathways.
This chapter begins with a brief survey of the nature of energy and the laws of thermodynamics. The participation of energy
in metabolism and the role of ATP as an energy currency is considered next. An introduction to the nature and function of enzymes follows. The chapter ends with an overview of metabolic
regulation, including an introduction to metabolic channeling and
the regulation of the activity of critical enzymes.
C
8.1
Energy and Work
Energy may be most simply defined as the capacity to do work
or to cause particular changes. Thus all physical and chemical
processes are the result of the application or movement of en-
Figure 8.1 The Flow of Carbon and Energy in an Ecosystem. This
diagram depicts the flow of energy and carbon in general terms. See text
for discussion.
ergy. Living cells carry out three major types of work, and all
are essential to life processes. Chemical work involves the synthesis of complex biological molecules required by cells from
much simpler precursors; energy is needed to increase the molecular complexity of a cell. Molecules and ions often must be
transported across cell membranes against an electro-chemical
gradient. For example, a molecule sometimes moves into a cell
even though its concentration is higher internally. Similarly a
solute may be expelled from the cell against a concentration
gradient. This process is transport work and requires energy
input in order to take up nutrients, eliminate wastes, and maintain ion balances. The third type of work is mechanical work,
perhaps the most familiar of the three. Energy is required to
change the physical location of organisms, cells, and structures
within cells.
The ultimate source of most biological energy is the visible
sunlight impinging on the earth’s surface. Light energy is
trapped by phototrophs during photosynthesis, in which it is absorbed by chlorophyll and other pigments and converted to
chemical energy. As noted in chapter 5, chemolithoautotrophs
derive energy by oxidizing inorganic compounds rather than obtaining it from light absorption. Chemical energy from photosynthesis and chemolithotrophy can then be used by photolithoautotrophs and chemolithoautotrophs to transform CO2
into biological molecules such as glucose (figure 8.1). Nutritional types (pp. 97–98)
The complex molecules manufactured by autotrophic organisms (both plant and microbial producers) serve as a carbon
source for chemoheterotrophs and other consumers that use complex organic molecules as a source of material and energy for
building their own cellular structures (it should be remembered
that autotrophs also use complex organic molecules). Chemoheterotrophs often employ O2 as an electron acceptor when oxidizing glucose and other organic molecules to CO2. This process,
in which O2 acts as the final electron acceptor and is reduced to
water, is called aerobic respiration. Much energy is released
8.2
5
9
N
4 3
N
6
8
O
–
O
O
P
O
O
–
O
P
O
O
–
P
O
O
CH2
1
2
155
ADP + Pi
NH2
N
7
The Laws of Thermodynamics
N
O
Aerobic respiration
Anaerobic respiration
Fermentation
Photosynthesis
Chemical work
Transport work
Mechanical work
–
ATP
OH
OH
Adenosine
Adenosine diphosphate (ADP)
Figure 8.3 The Cell’s Energy Cycle. ATP is formed from energy
made available during aerobic respiration, anaerobic respiration,
fermentation, and photosynthesis. Its breakdown to ADP and phosphate
(Pi) makes chemical, transport, and mechanical work possible.
Adenosine triphosphate (ATP)
(a)
8.2
(b)
Figure 8.2 Adenosine Triphosphate and Adenosine Diphosphate.
(a) Structure of ATP and ADP. The two red bonds are more easily
broken or have a high phosphate group transfer potential (see text). The
pyrimidine ring atoms have been numbered. (b) A model of ATP.
Carbon is in green; hydrogen in light blue; nitrogen in dark blue;
oxygen in red; and phosphorus in orange.
during this process. Thus, in the ecosystem, energy is trapped by
photoautotrophs and chemolithoautotrophs; some of this energy
subsequently flows to chemoheterotrophs when they use nutrients derived from autotrophs (figure 8.1; see also figure 28.32).
The CO2 produced during aerobic respiration can be incorporated
again into complex organic molecules during photosynthesis and
chemolithoautotrophy. Clearly the flow of carbon and energy in
the ecosystem is intimately related.
Cells must efficiently transfer energy from their energygenerating or trapping apparatus to the systems actually carrying
out work. That is, cells must have a practical form of energy currency. In living organisms the major currency is adenosine 5′triphosphate (ATP; figure 8.2). When ATP breaks down to
adenosine diphosphate (ADP) and orthophosphate (Pi), energy
is made available for useful work. Later, energy from photosynthesis, aerobic respiration, anaerobic respiration, and fermentation is used to resynthesize ATP from ADP and Pi. An energy cycle is created in the cell (figure 8.3). Fermentation (pp. 179–81);
Anaerobic respiration (pp. 190–91)
The Laws of Thermodynamics
To understand how energy is trapped or generated and how ATP
functions as an energy currency, some knowledge of the basic
principles of thermodynamics is required. The science of thermodynamics analyzes energy changes in a collection of matter
(e.g., a cell or a plant) called a system. All other matter in the universe is called the surroundings. Thermodynamics focuses on the
energy differences between the initial state and the final state of
a system. It is not concerned with the rate of the process. For instance, if a pan of water is heated to boiling, only the condition of
the water at the start and at boiling is important in thermodynamics, not how fast it is heated or on what kind of stove. Two important laws of thermodynamics must be understood. The first
law of thermodynamics says that energy can be neither created
nor destroyed. The total energy in the universe remains constant
although it can be redistributed. For example, many energy exchanges do occur during chemical reactions (e.g., heat is given off
by exothermic reactions and absorbed during endothermic reactions), but these heat exchanges do not violate the first law.
It is necessary to specify quantitatively the amount of energy
used in or evolving from a particular process, and two types of energy units are employed. A calorie (cal) is the amount of heat energy needed to raise one gram of water from 14.5 to 15.5°C. The
amount of energy also may be expressed in terms of joules (J),
the units of work capable of being done. One cal of heat is equivalent to 4.1840 J of work. One thousand calories or a kilocalorie
(kcal) is enough energy to boil 1.9 ml of water. A kilojoule is
enough energy to boil about 0.44 ml of water, or enable a person
weighing 70 kg to climb 35 steps. The joule is normally used by
chemists and physicists. Because biologists most often speak of
energy in terms of calories, this text will employ calories when
discussing energy changes.
Although it is true that energy is conserved in the universe, the
first law of thermodynamics does not account for many physical
and chemical processes. A simple example may help make this
clear. Suppose a full gas cylinder is connected to an empty one by
a tube with a valve (figure 8.4). If the valve is opened, gas flows
from the full to the empty cylinder until the gas pressure is equal on
156
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
Endergonic reactions
Exergonic reactions
A+B
Initial state
Keq
C+D
[C] [D]
[A] [B]
> 1 .0
A+B
Keq
∆ G°′ is negative.
C+D
[C] [D]
[A] [B]
< 1 .0
∆ G°′ is positive.
Figure 8.5 Go′ and Equilibrium. The relationship of Go′ to the
equilibrium of reactions. Note the differences between exergonic and
endergonic reactions.
Final state (equilibrium)
Figure 8.4 A Second Law Process. The expansion of gas into an
empty cylinder simply redistributes the gas molecules until
equilibrium is reached. The total number of molecules remains
unchanged.
The change in free energy has a definite, concrete relationship to the direction of chemical reactions. Consider the following simple reaction:
AB
both sides. Energy has not only been redistributed but also conserved. The expansion of gas is explained by the second law of
thermodynamics and a condition of matter called entropy. Entropy may be considered a measure of the randomness or disorder
of a system. The greater the disorder of a system, the greater is its
entropy. The second law states that physical and chemical
processes proceed in such a way that the randomness or disorder of
the universe (the system and its surroundings) increases to the maximum possible. Gas will always expand into an empty cylinder.
8.3
Free Energy and Reactions
The first and second laws can be combined in a useful equation,
relating the changes in energy that can occur in chemical reactions and other processes.
∆G ∆H T∆S
G is the change in free energy, H is the change in enthalpy, T is the temperature in Kelvin (°C 273), and S is the
change in entropy occurring during the reaction. The change in
enthalpy is the change in heat content. Cellular reactions occur
under conditions of constant pressure and volume. Thus the
change in enthalpy is about the same as the change in total energy
during the reaction. The free energy change is the amount of energy in a system available to do useful work at constant temperature and pressure. Therefore the change in entropy is a measure
of the proportion of the total energy change that the system cannot use in performing work. Free energy and entropy changes do
not depend on how the system gets from start to finish. A reaction
will occur spontaneously at constant temperature and pressure if
the free energy of the system decreases during the reaction or, in
other words, if G is negative. It follows from the equation that a
reaction with a large positive change in entropy will normally
tend to have a negative G value and therefore occur spontaneously. A decrease in entropy will tend to make G more positive and the reaction less favorable.
CD
If the molecules A and B are mixed, they will combine to
form the products C and D. Eventually C and D will become concentrated enough to combine and produce A and B at the same
rate as they are formed from A and B. The reaction is now at equilibrium: the rates in both directions are equal and no further net
change occurs in the concentrations of reactants and products.
This situation is described by the equilibrium constant (Keq), relating the equilibrium concentrations of products and substrates
to one another.
[C][D]
Keq ______
[A][B]
If the equilibrium constant is greater than one, the products are in
greater concentration than the reactants at equilibrium—that is,
the reaction tends to go to completion as written.
The equilibrium constant of a reaction is directly related to its
change in free energy. When the free energy change for a process is
determined at carefully defined standard conditions of concentration,
pressure, pH, and temperature, it is called the standard free energy
change (Go). If the pH is set at 7.0 (which is close to the pH of living cells), the standard free energy change is indicated by the symbol Go′. The change in standard free energy may be thought of as
the maximum amount of energy available from the system for useful
work under standard conditions. Using Go′ values allows one to
compare reactions without worrying about variations in the G due
to differences in environmental conditions. The relationship between
Go′ and Keq is given by the following equation:
∆Go´ 2.303RTlogKeq
R is the gas constant (1.9872 cal/mole-degree or 8.3145 J/moledegree), and T is the absolute temperature. Inspection of this equation shows that when G o′ is negative, the equilibrium constant is
greater than one and the reaction goes to completion as written. It
is said to be an exergonic reaction (figure 8.5). In an endergonic
reaction G o′ is positive and the equilibrium constant is less than
one. That is, the reaction is not favorable, and little product will be
formed at equilibrium under standard conditions. Keep in mind
8.5
Oxidation Reduction Reactions and Electron Carriers
that the G o′ value shows only where the reaction lies at equilibrium, not how fast the reaction reaches equilibrium.
1. What is energy and what kinds of work are carried out in a cell?
Describe the energy cycle and ATP’s role in it.
2. What is thermodynamics? Summarize the first and second laws.
Define free energy, entropy, and enthalpy.
3. How is the change in standard free energy related to the
equilibrium constant for a reaction? What are exergonic and
endergonic reactions?
8.4
Endergonic reaction alone
A+B
C+D
Endergonic reaction coupled to ATP breakdown
ATP
A+B
ADP + Pi
C+D
Figure 8.6 ATP as a Coupling Agent. The use of ATP to make
endergonic reactions more favorable. It is formed by exergonic
reactions and then used to drive endergonic reactions.
The Role of ATP in Metabolism
Many reactions in the cell are endergonic and will not proceed far
toward completion without outside assistance. One of ATP’s major roles is to drive such endergonic reactions more to completion.
ATP is a high-energy molecule. That is, it breaks down or hydrolyzes almost completely to the products ADP and Pi with a
G o′ of 7.3 kcal/mole.
ATP + H2O
ADP + Pi
In reference to ATP the term high-energy molecule does not mean
that there is a great deal of energy stored in a particular bond of
ATP. It simply indicates that the removal of the terminal phosphate goes to completion with a large negative standard free energy change, or the reaction is strongly exergonic. In other words,
ATP has a high phosphate group transfer potential; it readily
transfers its phosphate to water. The phosphate group transfer potential is defined as the negative of G o′ for the hydrolytic removal of phosphate. A molecule with a higher group transfer potential will donate phosphate to one with a lower potential.
Thus ATP is ideally suited for its role as an energy currency.
It is formed in energy-trapping and -generating processes such as
photosynthesis, fermentation, and aerobic respiration. In the cell’s
economy, exergonic ATP breakdown is coupled with various endergonic reactions to promote their completion (figure 8.6). In
other words ATP links energy-generating reactions, which liberate
free energy, with energy-using reactions, which require free energy input to proceed toward completion. Facilitation of chemical
work is the focus of the preceding example, but the same principles apply when ATP is coupled with endergonic processes involving transport work and mechanical work (figure 8.3).
8.5
157
Oxidation-Reduction Reactions
and Electron Carriers
Free energy changes are not only related to the equilibria of “regular” chemical reactions but also to the equilibria of oxidationreduction reactions. The release of energy normally involves
oxidation-reduction reactions. Oxidation-reduction (redox) reactions are those in which electrons move from a donor, the
reducing agent or reductant, to an electron acceptor, the
oxidizing agent or oxidant. By convention such a reaction is
written with the reductant to the right of the oxidant and the number (n) of electrons (e) transferred.
Oxidant ne–
reductant
The oxidant and reductant pair is referred to as a redox couple (table
8.1). When an oxidant accepts electrons, it becomes the reductant of
the couple. The equilibrium constant for the reaction is called the
standard reduction potential (E0) and is a measure of the tendency
of the reducing agent to lose electrons. The reference standard for
reduction potentials is the hydrogen system with an E′0 (the reduction potential at pH 7.0) of 0.42 volts or 420 millivolts.
2H+ + 2e–
H2
In this reaction each hydrogen atom provides one proton (H)
and one electron (e).
The reduction potential has a concrete meaning. Redox couples with more negative reduction potentials will donate electrons
to couples with more positive potentials and greater affinity for
electrons. Thus electrons will tend to move from reductants at the
top of the list in table 8.1 to oxidants at the bottom because they
have more positive potentials. This may be expressed visually in
the form of an electron tower in which the most negative reduction potentials are at the top (figure 8.7). Electrons move from
donors to acceptors down the potential gradient or fall down the
tower to more positive potentials. Consider the case of the electron carrier nicotinamide adenine dinucleotide (NAD). The
NAD/NADH couple has a very negative E′0 and can therefore
give electrons to many acceptors, including O2.
NAD+ + 2H+ + 2e–
NADH + H+
E0́
= –0.32 volts
1/2O2 + 2H+ + 2e–
H2O
E0́
= +0.82 volts
Because NAD /NADH is more negative than 1/2 O2/H2O, electrons will flow from NADH (the reductant) to O2 (the oxidant) as
shown in figure 8.7.
NADH + H+ + 1/2O2
H2O + NAD+
When electrons move from a reductant to an acceptor with a
more positive redox potential, free energy is released. The G o′ of
the reaction is directly related to the magnitude of the difference
158
Chapter 8
Table 8.1
Metabolism: Energy, Enzymes, and Regulation
Redox Couple
E´0 (Volts)
2H+ 2e–
H2
ferredoxin (Fe2+)
Ferredoxin(Fe3+) e–
NAD(P)+ H+ 2e–
NAD(P)H
S + 2H+ 2e–
H2S
Acetaldehyde 2H+ 2e–
ethanol
Pyruvate– 2H+ + 2e–
lactate2–
FAD + 2H+ 2e–
FADH2
Oxaloacetate2– 2H+ 2e–
malate2–
Fumarate2– 2H+ 2e–
succinate2–
Cytochrome b (Fe3+) e–
cytochrome b (Fe2+)
Ubiquinone 2H+ 2e–
ubiquinone H2
Cytochrome c (Fe3+) e–
cytochrome c (Fe2+)
NO3– 2H+ 2e–
NO2– H2O
NO2– 8H+ 6e–
NH4+ 2H2O
Fe2+
Fe3+ e–
O2 4H+ 4e–
2H2O
E0′ (Volts)
Better
electron donors
Selected Biologically Important
Redox Couples
–0.5
2H+/H2 [–0.42]
–0.4
a
–0.42
–0.42
–0.32
–0.274
–0.197
–0.185
–0.18b
–0.166
0.031
0.075
0.10
0.254
0.421
0.44
0.771
0.815
+
NAD /NADH [–0.32]
FAD/FADH2 [–0.18]
Fumarate/succinate [0.031]
CoQ/CoQH2 [0.10]
+0.1
Cyt c (Fe3+ )/Cyt c (Fe2+ ) [0.254]
+0.3
NO3–/NO2– [0.421]
NADH + H+ + 1/2O2
H2O + NAD+
(∆E0′ = 1.14 V)
+0.4
+0.5
+0.6
+0.7
Fe3+/Fe2+ [0.771]
1
/2O2/ H2O [0.815]
E´0 is the standard reduction potential at pH 7.0.
The value for FAD/FADH2 applies to the free cofactor because it can vary considerably when bound
to an apoenzyme.
+0.8
+0.9
Better
electron acceptors
in which n is the number of electrons transferred and F is the
Faraday constant (23,062 cal/mole-volt or 96,494 J/mole-volt).
For every 0.1 volt change in E′0, there is a corresponding 4.6
kcal change in G o′ when a two-electron transfer takes place.
This is similar to the relationship of Go′ and Keq in other chemical reactions—the larger the equilibrium constant, the greater
the G o′. The difference in reduction potentials between
NAD/NADH and 1/2O2/H2O is 1.14 volts, a large E′0 value.
When electrons move from NADH to O2 during aerobic respiration, a large amount of free energy is made available to synthesize ATP (figure 8.8). If energy is released when electrons
flow from negative to positive reduction potentials, then an input of energy is required to move electrons in the opposite direction, from more positive to more negative potentials. This is
precisely what occurs during photosynthesis (figure 8.8). Light
energy is trapped and used to move electrons from water to the
electron carrier nicotinamide adenine dinucleotide phosphate (NADP).
The cycle of energy flow discussed earlier and illustrated in
figure 8.1 can be understood from a different perspective, if the
preceding concept is kept in mind. Photosynthetic organisms
capture light energy and use it to move electrons from water (and
other electron donors such as H2S) to electron acceptors, such as
2e–
0.0
+0.2
b
∆Go´ nF∆E´0
–0.2
–0.1
a
between the reduction potentials of the two couples (E′0). The
larger the E′0, the greater the amount of free energy made available, as is evident from the equation
–0.3
+1.0
Figure 8.7 Electron Movement and Reduction Potentials. The
vertical electron tower in this illustration has the most negative
reduction potentials at the top. Electrons will spontaneously move from
donors higher on the tower (more negative potentials) to acceptors
lower on the tower (more positive potentials). That is, the reductant is
always higher on the tower than the oxidant. For example, NADH will
donate electrons to oxygen and form water in the process. Some typical
donors and acceptors are shown on the left, and their redox potentials
are given in brackets.
More
negative E′0
NADH
2e–
More
positive E′0
1/2
NADP
ATP
O2
Aerobic respiration
Light
energy
+
2e–
H 2O
Oxygenic photosynthesis
Figure 8.8 Energy Flow in Metabolism. Examples of the
relationship between electron flow and energy in metabolism.
Oxygen and NADP serve as electron acceptors for NADH and
water, respectively.
8.5
H
O
C
O
HO
S
H
NH2
O
C
+
S +
P
O
CH2
N
N+
O
Ribose
Reduced
substrate
P
O
OH
N
+
Oxidized
substrate
R
NAD
NH2
+ H+
H
O
HO
C
Nicotinamide
H H
O
H
NH2
159
Oxidation Reduction Reactions and Electron Carriers
+
R
NADH
(b)
OH
NH2
N
O
N
Adenine
CH2
O
N
N
Nicotinamide
unit
Ribose
OH OH
Ribose
unit
NADP has a phosphate here.
(a)
Figure 8.9 The Structure and Function of NAD. (a) The structure
of NAD and NADP. NADP differs from NAD in having an extra
phosphate on one of its ribose sugar units. (b) NAD can accept
electrons and a hydrogen from a reduced substrate (SH2). These are
carried on the nicotinamide ring. (c) Model of NAD when bound to
the enzyme lactate dehydrogenase.
NADP, that have more negative reduction potentials. These
electrons can then flow back to more positive acceptors and provide energy for ATP production during photosynthesis. Photoautotrophs use ATP and NADPH to synthesize complex molecules from CO2 (see section 10.2). Chemoheterotrophs also
make use of energy released during the movement of electrons
by oxidizing complex nutrients during respiration to produce
NADH. NADH subsequently donates its electrons to O2, and the
energy released during electron transfer is trapped in the form of
ATP. The energy from sunlight is made available to all living organisms because of this relationship between electron flow and
energy. Photosynthesis (pp. 195–201); Respiration and electron transport
(pp. 184–89)
Electron transport is important in aerobic respiration, anaerobic respiration, chemolithotrophy, and photosynthesis. Electron
movement in cells requires the participation of carriers such as
NAD and NADP, both of which can transport electrons between different locations. The nicotinamide ring of NAD and
NADP (figure 8.9) accepts two electrons and one proton from a
donor, while a second proton is released. There are several other
electron carriers of importance in microbial metabolism (table
8.1), and they carry electrons in a variety of ways. Flavin adenine
dinucleotide (FAD) and flavin mononucleotide (FMN) bear
two electrons and two protons on the complex ring system shown
in figure 8.10. Proteins bearing FAD and FMN are often called
flavoproteins. Coenzyme Q (CoQ) or ubiquinone is a quinone
that transports electrons and protons in many respiratory electron
transport chains (figure 8.11). Cytochromes and several other
Adenine
unit
Ribose
unit
Pyrophosphate
unit
(c)
carriers use iron atoms to transport electrons by reversible oxidation and reduction reactions.
Fe3+ (ferric iron) e–
Fe2+ (ferrous iron)
In the cytochromes these iron atoms are part of a heme group (figure 8.12) or other similar iron-porphyrin rings. Several different
cytochromes, each of which consists of a protein and an ironporphyrin ring, are a prominent part of respiratory electron transport chains. Some iron containing electron-carrying proteins lack a
heme group and are called nonheme iron proteins. Ferredoxin is
a nonheme iron protein active in photosynthetic electron transport
and several other electron transport processes. Even though its iron
atoms are not bound to a heme group, they still undergo reversible
oxidation and reduction reactions. Although all the previously discussed molecules function in electron transport chains, some bear
two electrons (NAD, FAD, and CoQ) while others carry only one
electron at a time (cytochromes and nonheme iron proteins). This
difference in the number of electrons carried is of great importance
in the operation of electron transport chains (see pp. 184–87).
1. Why is ATP called a high-energy molecule? What is its role in the
cell and how does it fulfill this role?
2. Write a generalized equation for a redox reaction. Define
reductant, oxidant, and standard reduction potential.
3. How is the direction of electron flow between redox couples related
to the standard reduction potential and the release of free energy?
Name and briefly describe the major electron carriers found in cells.
160
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
H
O
N
CH3
NH
CH3
N
O
N
H
O
N
CH3
NH
+
2e– + 2H +
Isoalloxazine ring
CH3
N
O
N
CH2
H
C
OH
H
C
OH
H
C
OH
CH2
Figure 8.10 The Structure and Function of FAD.
The vitamin riboflavin is composed of the
isoalloxazine ring and its attached ribose sugar. FMN is
riboflavin phosphate. The portion of the ring directly
involved in oxidation-reduction reactions is in color.
O
N
O
P
O
O
O
–
O
(CH2
CH
C
H
CH2)n
H
C
H
O
CH3
HC
CH3
CH3
(CH2
O
CH
(CH2)3
OH
CH
C
CH2)n
N
Adenine
H
O
Figure 8.11 The Structure and Function of Coenzyme Q or
Ubiquinone. The length of the side chain varies among organisms
from n 6 to n 10.
CH3
CH
(CH2)3
H
C
C
N
N
C
N
C
Fe
C
C
CH3
CH3
C
C
O
+
N
O
C
C
O
CH3
C
CH3
2H + 2e–+
CH2
CH
C
O H
CH3
O
–
CH3
CH2
CH3
O
P
OH OH
Ribose
CH3
CH3
N
O
O H
CH3
NH2
Ribose
N
C
C
C
C
C
CH2
H
CH2
C
CH
C
H
C
H
CH2
CH2
COOH
COOH
CH2
Figure 8.12 The Structure of Heme. Heme is composed of a
porphyrin ring and an attached iron atom. It is the nonprotein
component of many cytochromes. The iron atom alternatively accepts
and releases an electron.
8.6
Table 8.2
Enzymes
Enzyme Classification
Type of Enzyme
Reaction Catalyzed by Enzyme
Example of Reaction
Oxidoreductase
Oxidation-reduction reactions
Transferase
Reactions involving the transfer of groups
between molecules
Hydrolase
Hydrolysis of molecules
Lyase
Removal of groups to form double bonds or
addition of groups to double bonds
Lactate dehydrogenase:
Pyruvate + NADH + H+
lactate + NAD+
Aspartate carbamoyltransferase:
Aspartate + carbamoylphosphate
carbamoylaspartate + phosphate
Glucose-6-phosphatase:
Glucose 6-phosphate + H2O → glucose + Pi
Fumarate hydratase:
L-malate
fumarate + H2O
c
c + x
y
x
y
c
c
Isomerase
Reactions involving isomerizations
Ligase
Joining of two molecules using ATP energy
(or that of other nucleoside triphosphates)
8.6
161
Enzymes
Recall that an exergonic reaction is one with a negative G o′ and
an equilibrium constant greater than one. An exergonic reaction
will proceed to completion in the direction written (that is, toward
the right of the equation). Nevertheless, one often can combine
the reactants for an exergonic reaction with no obvious result,
even though products should be formed. It is precisely in these reactions that enzymes play their part.
Structure and Classification of Enzymes
Enzymes may be defined as protein catalysts that have great
specificity for the reaction catalyzed and the molecules acted on.
A catalyst is a substance that increases the rate of a chemical reaction without being permanently altered itself. Thus enzymes
speed up cellular reactions. The reacting molecules are called
substrates, and the substances formed are the products. Protein
structure and properties (appendix I)
Many enzymes are indeed pure proteins. However, many
enzymes consist of a protein, the apoenzyme, and also a nonprotein component, a cofactor, required for catalytic activity.
The complete enzyme consisting of the apoenzyme and its cofactor is called the holoenzyme. If the cofactor is firmly attached to the apoenzyme it is a prosthetic group. Often the cofactor is loosely attached to the apoenzyme. It can even
dissociate from the enzyme protein after products have been
formed and carry one of these products to another enzyme (figure 8.13). Such a loosely bound cofactor is called a coenzyme.
For example, NAD is a coenzyme that carries electrons within
the cell. Many vitamins that humans require serve as coenzymes
or as their precursors. Niacin is incorporated into NAD and riboflavin into FAD. Metal ions may also be bound to apoenzymes and act as cofactors.
Alanine racemase:
L-alanine
D-alanine
Glutamine synthetase:
Glutamate + NH3 + ATP → glutamine + ADP + Pi
E1
A
C
B
C—X
P
E2
Q
Figure 8.13 Coenzymes as Carriers. The function of a coenzyme
in carrying substances around the cell. Coenzyme C participates with
enzyme E1 in the conversion of A to product B. During the reaction, it
acquires X from the substrate A. The coenzyme can donate X to
substrate P in a second reaction. This will convert it back to its original
form, ready to accept another X. The coenzyme is not only
participating in both reactions, but is also transporting X to various
points in the cell.
Despite the large number and bewildering diversity of enzymes present in cells, they may be placed in one of six general
classes (table 8.2). Enzymes usually are named in terms of the substrates they act on and the type of reaction catalyzed. For example,
lactate dehydrogenase (LDH) removes hydrogens from lactate.
Lactate + NAD+
LDH
pyruvate + NADH + H+
Lactate dehydrogenase can also be given a more complete and detailed name, L-lactate: NAD oxidoreductase. This name describes
the substrates and reaction type with even more precision.
The Mechanism of Enzyme Reactions
It is important to keep in mind that enzymes increase the rates of reactions but do not alter their equilibrium constants. If a reaction is
endergonic, the presence of an enzyme will not shift its equilibrium
162
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
Substrates
AB
‡
Free energy
Ea
Active site
Enzyme
A+B
∆G°′
C+D
Enzyme-substrate complex
Progress of the reaction
Product
Figure 8.14 Enzymes Lower the Energy of Activation. This figure
traces the course of a chemical reaction in which A and B are converted
to C and D. The transition-state complex is represented by AB, and
the activation energy required to reach it, by Ea. The colored line
represents the course of the reaction in the presence of an enzyme.
Note that the activation energy is much lower in the enzyme-catalyzed
reaction.
so that more products can be formed. Enzymes simply speed up the
rate at which a reaction proceeds toward its final equilibrium.
How do enzymes catalyze reactions? Although a complete
answer would be long and complex, some understanding of the
mechanism can be gained by considering the course of a normal
exergonic chemical reaction.
A+B
C+D
When molecules A and B approach each other to react, they form a
transition-state complex, which resembles both the substrates and
the products (figure 8.14). The activation energy is required to
bring the reacting molecules together in the correct way to reach the
transition state. The transition-state complex can then decompose to
yield the products C and D. The difference in free energy level between reactants and products is Go′. Thus the equilibrium in our
example will lie toward the products because G o′ is negative (i.e.,
the products are at a lower energy level than the substrates).
Clearly A and B will not be converted to C and D in figure
8.14 if they are not supplied with an amount of energy equivalent
to the activation energy. Enzymes accelerate reactions by lowering the activation energy; therefore more substrate molecules will
have sufficient energy to come together and form products. Even
though the equilibrium constant (or Go′) is unchanged, equilibrium will be reached more rapidly in the presence of an enzyme
because of this decrease in the activation energy.
Researchers have expended much effort in discovering how
enzymes lower the activation energy of reactions, and the process
is becoming clearer. Enzymes bring substrates together at a special
place on their surface called the active site or catalytic site to form
an enzyme-substrate complex (figures 8.15, 8.16; see also
AI.19). The enzyme can interact with a substrate in two general
Figure 8.15 Enzyme Function. The formation of the enzymesubstrate complex and its conversion to products is shown.
ways. It may be rigid and shaped to precisely fit the substrate so that
the correct substrate binds specifically and is positioned properly
for reaction. This mechanism is referred to as the lock-and-key
model. An enzyme also may change shape when it binds the substrate so that the active site surrounds and precisely fits the substrate. This has been called the induced fit model and is used by
hexokinase and many other enzymes (figure 8.16). The formation
of an enzyme-substrate complex can lower the activation energy in
many ways. For example, by bringing the substrates together at the
active site, the enzyme is, in effect, concentrating them and speeding up the reaction. An enzyme does not simply concentrate its substrates, however. It also binds them so that they are correctly oriented with respect to each other in order to form a transition-state
complex. Such an orientation lowers the amount of energy that the
substrates require to reach the transition state. These and other catalytic site activities speed up a reaction hundreds of thousands of
times, even though microorganisms grow between 20°C and approximately 113°C. These temperatures are not high enough to favor most organic reactions in the absence of enzyme catalysis, yet
cells cannot survive at the high temperatures used by an organic
chemist in routine organic syntheses. Enzymes make life possible
by accelerating specific reactions at low temperatures.
The Effect of Environment on Enzyme Activity
Enzyme activity varies greatly with changes in environmental
factors, one of the most important being the substrate concentration. As will be emphasized later, substrate concentrations are
8.6
(a)
Enzymes
163
(b)
Figure 8.16 An Example of Enzyme-Substrate Complex Formation. (a) A space-filling model of yeast
hexokinase and its substrate glucose (purple). The active site is in the cleft formed by the enzyme’s small lobe
(green) and large lobe (gray). (b) When glucose binds to form the enzyme-substrate complex, hexokinase
changes shape and surrounds the substrate.
Vmax
Velocity
usually low within cells. At very low substrate concentrations, an
enzyme makes product slowly because it seldom contacts a substrate molecule. If more substrate molecules are present, an enzyme binds substrate more often, and the reaction velocity (usually expressed in terms of the rate of product formation) is greater
than at a lower substrate concentration. Thus the rate of an
enzyme-catalyzed reaction increases with substrate concentration
(figure 8.17). Eventually further increases in substrate concentration do not result in a greater reaction velocity because the
available enzyme molecules are binding substrate and converting
it to product as rapidly as possible. That is, the enzyme is saturated with substrate and operating at maximal velocity (Vmax).
The resulting substrate concentration curve is a hyperbola (figure
8.17). It is useful to know the substrate concentration an enzyme
needs to function adequately. Usually the Michaelis constant
(Km), the substrate concentration required for the enzyme to
achieve half maximal velocity, is used as a measure of the apparent affinity of an enzyme for its substrate. The lower the Km value,
the lower the substrate concentration at which an enzyme catalyzes its reaction.
Enzymes also change activity with alterations in pH and temperature (figure 8.18). Each enzyme functions most rapidly at a
specific pH optimum. When the pH deviates too greatly from an
enzyme’s optimum, activity slows and the enzyme may be damaged. Enzymes likewise have temperature optima for maximum
activity. If the temperature rises too much above the optimum, an
enzyme’s structure will be disrupted and its activity lost. This
phenomenon, known as denaturation, may be caused by
Vmax
2
v=
Vmax•S
Km + S
Km
Substrate concentration
Km = the substrate concentration required by the
enzyme to operate at half its maximum
velocity
Vmax = the rate of product formation when the
enzyme is saturated with substrate and
operating as fast as possible
Figure 8.17 Michaelis-Menten Kinetics. The dependence of
enzyme activity upon substrate concentration. This substrate curve fits
the Michaelis-Menten equation given in the figure, which relates
reaction velocity (v) to the substrate concentration (S) using the
maximum velocity and the Michaelis constant (Km).
Metabolism: Energy, Enzymes, and Regulation
Figure 8.18 pH, Temperature, and Enzyme
Activity. The variation of enzyme activity with
changes in pH and temperature. The ranges in pH
and temperature are only representative. Enzymes
differ from one another with respect to the location
of their optima and the shape of their pH and
temperature curves.
Velocity
Chapter 8
Velocity
164
5
7
10
pH
COOH
COOH
CH2
CH2
COOH
Succinic acid
CH2
COOH
Malonic acid
Figure 8.19 Competitive Inhibition of Succinate Dehydrogenase. A
comparison of succinic acid and the competitive inhibitor, malonic acid.
The colored atoms indicate the parts of the two molecules that differ.
extremes of pH and temperature or by other factors. The pH and
temperature optima of a microorganism’s enzymes often reflect
the pH and temperature of its habitat. Not surprisingly bacteria
growing best at high temperatures often have enzymes with high
temperature optima and great heat stability. Temperature and growth
10
30
50
70
Temperature (°C)
Inhibitors also can affect enzyme activity by binding to the
enzyme at some location other than at the active site. This alters
the enzyme’s shape, rendering it inactive or less active. These inhibitors are often called noncompetitive because they do not directly compete with the substrate. Heavy metal poisons like mercury frequently are noncompetitive inhibitors of enzymes.
1. What is an enzyme and how does it speed up reactions? How are
enzymes named? Define apoenzyme, holoenzyme, cofactor,
coenzyme, prosthetic group, active or catalytic site, and activation
energy.
2. How does enzyme activity change with substrate concentration,
pH, and temperature? Define the terms Michaelis constant,
maximum velocity, and denaturation.
3. What are competitive and noncompetitive inhibitors and how do
they inhibit enzymes?
(pp. 125–27); Heat stable enzymes in biotechnology (p. 626)
Enzyme Inhibition
Microorganisms can be poisoned by a variety of chemicals, and
many of the most potent poisons are enzyme inhibitors. A competitive inhibitor directly competes with the substrate at an enzyme’s
catalytic site and prevents the enzyme from forming product. A classic example of this behavior is seen with the enzyme succinate dehydrogenase, which catalyzes the oxidation of succinate to fumarate
in the tricarboxylic acid cycle (see section 9.4). Malonic acid is an
effective competitive inhibitor of succinate dehydrogenase because
it so closely resembles succinate, the normal substrate (figure 8.19).
After malonate binds to the enzyme, it cannot be oxidized and the
formation of fumarate is blocked. Competitive inhibitors usually resemble normal substrates, but they cannot be converted to products.
Competitive inhibitors are important in the treatment of
many microbial diseases. Sulfa drugs like sulfanilamide resemble
p-aminobenzoate, a molecule used in the formation of the coenzyme folic acid. The drugs compete with p-aminobenzoate for the
catalytic site of an enzyme involved in folic acid synthesis. This
blocks the production of folic acid and inhibits bacterial growth
(see section 35.6). Humans are not harmed because they cannot
synthesize folic acid and must obtain it in their diet. Destruction
of microorganisms by physical and chemical agents (chapter 7)
8.7
The Nature and Significance
of Metabolic Regulation
The task of the regulatory machinery is exceptionally complex and
difficult. Pathways must be regulated and coordinated so effectively that all cell components are present in precisely the correct
amounts. Furthermore, a microbial cell must be able to respond effectively to environmental changes by using those nutrients present at the moment and by switching on new catabolic pathways
when different nutrients become available. Because all chemical
components of a cell usually are not present in the surroundings,
microorganisms also must synthesize unavailable components and
alter biosynthetic activity in response to changes in nutrient availability. The chemical composition of a cell’s surroundings is constantly changing, and these regulatory processes are dynamic and
continuously responding to altered conditions.
Regulation is essential for the cell to conserve microbial energy and material and to maintain metabolic balance. If a particular energy source is unavailable, the enzymes required for its use
are not needed and their further synthesis is a waste of carbon, nitrogen, and energy. Similarly it would be extremely wasteful for
a microorganism to synthesize the enzymes required to manufacture a certain end product if that end product were already pres-
8.9
ent in adequate amounts. Thus both catabolism and anabolism are
regulated in such a way as to maximize efficiency of operation.
Catabolism and anabolism (p. 173)
The drive to maintain balance and conserve energy and material is evident in the regulatory responses of a bacterium like E.
coli. If the bacterium is grown in a very simple medium containing only glucose as a carbon and energy source, it will synthesize
the required cell components in balanced amounts. Addition of a
biosynthetic end product (the amino acid tryptophan, for example) to the medium will result in the immediate inhibition of the
pathway synthesizing that end product; synthesis of the pathway’s enzymes also will slow or cease. If E. coli is transferred to
medium containing only the sugar lactose, it will synthesize the
enzymes required for catabolism of this nutrient. In contrast,
when E. coli grows in a medium possessing both glucose and lactose, glucose (the sugar supporting most rapid growth) is catabolized first. The culture will use lactose only after the glucose supply has been exhausted.
The flow of carbon through a pathway may be regulated in
three major ways.
1. The localization of metabolites and enzymes in different
parts of a cell, a phenomenon called metabolic channeling,
influences pathway activity.
2. Critical enzymes often are directly stimulated or inhibited
to alter pathway activity rapidly.
3. The number of enzyme molecules also may be controlled.
The more catalyst molecules present, the greater the
pathway’s activity. In bacteria regulation is usually exerted
at the level of transcription. Control of mRNA synthesis is
slower than direct regulation of enzyme activity but does
result in the saving of much energy and raw material
because enzymes are not synthesized when not required.
Each of these mechanisms is described in detail. This chapter introduces the first two: metabolic channeling and direct control of
enzyme activity. The discussion of gene expression regulation
follows a description of DNA, RNA, and protein synthesis in
chapters 11 and 12. Regulation of gene expression (pp. 275–83)
8.8
Control of Enzyme Activity
165
tion of NAD between the two compartments will then determine
the relative activity of these competing pathways, and the pathway with access to the most NAD will be favored.
Channeling also occurs within compartments such as the cytoplasmic matrix. The matrix is a structured dense material with
many subcompartments. In eucaryotes it also is subdivided by the
endoplasmic reticulum and cytoskeleton (see chapter 4). Metabolites and coenzymes do not diffuse rapidly in such an environment,
and metabolite gradients will build up near localized enzymes or
enzyme systems. This occurs because enzymes at a specific site
convert their substrates to products, resulting in decreases in the
concentration of one or more metabolites and increases in others.
For example, product concentrations will be high near an enzyme
and decrease with increasing distance from it.
Channeling can generate marked variations in metabolite concentrations and therefore directly affect enzyme activity. Substrate
levels are generally around 103 moles/liter (M) to 106 M or even
lower. Thus they may be in the same range as enzyme concentrations and equal to or less than the Michaelis constants (Km) of many
enzymes. Under these conditions the concentration of an enzyme’s
substrate may control its activity because the substrate concentration is in the rising portion of the hyperbolic substrate saturation
curve (figure 8.20). As the substrate level increases, it is converted
to product more rapidly; a decline in substrate concentration automatically leads to lower enzyme activity. If two enzymes in different pathways use the same metabolite, they may directly compete
for it. The pathway winning this competition—the one with the enzyme having the lowest Km value for the metabolite—will operate
closer to full capacity. Thus channeling within a cell compartment
can regulate and coordinate metabolism through variations in
metabolite and coenzyme levels. Enzyme kinetics and the substrate saturation curve (pp. 162–63)
1. Give three ways in which the flow of carbon through a pathway
may be regulated.
2. Define the terms metabolic channeling and compartmentation.
How are they involved in the regulation of metabolism?
Metabolic Channeling
8.9
One of the most common channeling mechanisms is that of compartmentation, the differential distribution of enzymes and
metabolites among separate cell structures or organelles. Compartmentation is particularly important in eucaryotic microorganisms with their many membrane-bound organelles. For example,
fatty acid oxidation is located within the mitochondrion, whereas
fatty acid synthesis occurs in the cytoplasmic matrix. The
periplasm in procaryotes can also be considered an example of
compartmentation (see p. 55). Compartmentation makes possible
the simultaneous, but separate, operation and regulation of similar pathways. Furthermore, pathway activities can be coordinated
through regulation of the transport of metabolities and coenzymes between cell compartments. Suppose two pathways in different cell compartments require NAD for activity. The distribu-
Control of Enzyme Activity
Adjustment of the activity of regulatory enzymes controls the
functioning of many metabolic pathways. This section describes
these enzymes and discusses their role in regulating pathway
activity.
Allosteric Regulation
Usually regulatory enzymes are allosteric enzymes. The activity
of an allosteric enzyme is altered by a small molecule known as
an effector or modulator. The effector binds reversibly by noncovalent forces to a regulatory site separate from the catalytic
site and causes a change in the shape or conformation of the enzyme (figure 8.21). The activity of the catalytic site is altered as
166
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
Vmax
Velocity
Catalytic
site
Regulatory
site
Effector or modulator
Vmax
2
Substrate
A
Km
B
[Substrate]
Figure 8.20 Control of Enzyme Activity by Substrate
Concentration. An enzyme-substrate saturation curve with the
Michaelis constant (Km) and the velocity equivalent to half the
maximum velocity (Vmax) indicated. The initial velocity of the reaction
(v) is plotted against the substrate concentration [Substrate]. The
maximum velocity is the greatest velocity attainable with a fixed
amount of enzyme under defined conditions. When the substrate
concentration is equal to or less than the Km , the enzyme’s activity will
vary almost linearly with the substrate concentration. Suppose the
substrate increases in concentration from level A to B. Because these
concentrations are in the range of the Km , a significant increase in
enzyme activity results. A drop in concentration from B to A will lower
the rate of product formation.
a result. A positive effector increases enzyme activity, whereas a
negative effector decreases activity or inhibits the enzyme. These
changes in activity often result from alterations in the apparent
affinity of the enzyme for its substrate, but changes in maximum
velocity also can occur.
The kinetic characteristics of nonregulatory enzymes show
that the Michaelis constant (Km) is the substrate concentration required for an enzyme to operate at half its maximal velocity. This
constant applies only to hyperbolic substrate saturation curves,
not to the sigmoidal curves often seen with allosteric enzymes
(figure 8.23). The substrate concentration required for half maximal velocity with allosteric enzymes having sigmoidal substrate
curves is called the [S]0.5 or K0.5 value.
One of the best-studied allosteric regulatory enzymes is the
aspartate carbamoyltransferase (ACTase) from E. coli. The enzyme catalyzes the condensation of carbamoyl phosphate with
aspartate to form carbamoylaspartate (figure 8.22). ACTase catalyzes the rate-determining reaction of the pyrimidine biosynthetic pathway in E. coli. The substrate saturation curve is sigmoidal when the concentration of either substrate is varied
Figure 8.21 Allosteric Regulation. The structure and function of an
allosteric enzyme. In this example the effector or modulator first binds
to a separate regulatory site and causes a change in enzyme
conformation that results in an alteration in the shape of the active site.
The active site can now more effectively bind the substrate. This
effector is a positive effector because it stimulates substrate binding
and catalytic activity.
(figure 8.23). The enzyme has more than one active site, and the
binding of a substrate molecule to an active site increases the
binding of substrate at the other sites. In addition, cytidine
triphosphate (CTP), an end product of pyrimidine biosynthesis,
inhibits the enzyme and the purine ATP activates it. Both effectors alter the K0.5 value of the enzyme but not its maximum velocity. CTP inhibits by increasing K0.5 or by shifting the substrate saturation curve to higher values. This allows the enzyme
to operate more slowly at a particular substrate concentration
when CTP is present. ATP activates by moving the curve to
lower substrate concentration values so that the enzyme is maximally active over a wider substrate concentration range. Thus
when the pathway is so active that the CTP concentration rises
too high, ACTase activity decreases and the rate of end product
formation slows. In contrast, when the purine end product ATP
increases in concentration relative to CTP, it stimulates CTP synthesis through its effects on ACTase. Pyrimidine and purine biosynthesis (pp. 216–18)
E. coli aspartate carbamoyltransferase provides a clear example of separate regulatory and catalytic sites in allosteric en-
8.9
Control of Enzyme Activity
O
L-Glutamine
–
Carbamoyl
phosphate
synthetase
NH2
O
+ HCO3 + 2ATP
–O
CH2
+
O
–
–O
O
C
C
P
O–
Carbamoyl
phosphate
H2N
–O
Aspartate
carbamoyltransferase
+
CH
O
167
NH2
CH2
C
CH
+ Pi
–
O
COO–
C
N
H
COO–
Carbamoylaspartate
Aspartate
ATP
CTP
Uridine monophosphate (UMP)
Figure 8.22 ACTase Regulation. The aspartate carbamoyltransferase reaction and its role in the regulation of
pyrimidine biosynthesis. The end product CTP inhibits its activity () while ATP activates the enzyme ().
Carbamoyl phosphate synthetase is also inhibited by pathway end products such as UMP.
Vmax
+ATP
Velocity
+CTP
Vmax
2
zymes. The enzyme is a large protein composed of two catalytic
subunits and three regulatory subunits (figure 8.24a). The catalytic subunits contain only catalytic sites and are unaffected by
CTP and ATP. Regulatory subunits do not catalyze the reaction
but do possess regulatory sites to which CTP and ATP bind. When
these effectors bind to the complete enzyme, they cause conformational changes in the regulatory subunits and subsequently in
the catalytic subunits and their catalytic sites. The enzyme can
change reversibly between a less active T form and a more active
R form (figure 8.24b,c). Thus the regulatory site influences a catalytic site about 6.0 nm distant.
Covalent Modification of Enzymes
K0.5
K0.5
K0.5
[Substrate]
Figure 8.23 The Kinetics of E. coli Aspartate
Carbamoyltransferase. CTP, a negative effector, increases the K0.5
value while ATP, a positive effector, lowers the K0.5. The Vmax remains
constant.
Regulatory enzymes also can be switched on and off by reversible
covalent modification. Usually this occurs through the addition
and removal of a particular group, one form of the enzyme being
more active than the other. For example, glycogen phosphorylase
of the bread mold Neurospora crassa exists in phosphorylated and
dephosphorylated forms called phosphorylase a and phosphorylase b, respectively (figure 8.25). Phosphorylase b is inactive
168
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
Catalytic subunit
Approximate location
of the catalytic site
Regulatory
subunit
Regulator y sites
(a)
(c)
(b)
Figure 8.24 The Structure and Regulation of E. coli Aspartate Carbamoyltransferase. (a) A schematic
diagram of the enzyme showing the six catalytic polypeptide chains (blue), the six regulatory chains (orange),
and the catalytic and regulatory sites. The enzyme is viewed from the top. Each catalytic subunit contains three
catalytic chains, and each regulatory subunit has two chains. (b) The less active T state of ACTase viewed from
the side. (c) The more active R state of ACTase. The regulatory subunits have rotated and pushed the catalytic
subunits apart.
Phosphorylase b
(inactive)
Pi
ATP
H2O
ADP
P
Phosphorylase a
(active)
(Glucose)n
+
Pi
(Glucose)n–1
+
Glucose –1– P
Figure 8.25 Reversible Covalent Modification of Glycogen
Phosphorylase. The active form, phosphorylase a, is produced by
phosphorylation and is inactivated when the phosphate is removed
hydrolytically to produce inactive phosphorylase b.
because its required activator AMP is usually not present at sufficiently high levels. Phosphorylase a, the phosphorylated form, is
active even without AMP. Glycogen phosphorylase is stimulated
by phosphorylation of phosphorylase b to produce phosphorylase
a. The attachment of phosphate changes the enzyme’s conformation to an active form. Phosphorylation and dephosphorylation are
catalyzed by separate enzymes, which also are regulated. Phosphorylase and glycogen degradation (p. 192)
Enzymes can be regulated through the attachment of groups
other than phosphate. One of the most intensively studied regulatory enzymes is E. coli glutamine synthetase, a large, complex enzyme existing in two forms (figure 8.26). When an adenylic acid
residue is attached to each of its 12 subunits forming an adenyly-
(a)
(b)
Figure 8.26 The Structure of E. coli Glutamine Synthetase. The
enzyme contains 12 subunits in the shape of a hexagonal prism. For
clarity the subunits are colored alternating green and blue. Each of the
six catalytic sites has a pair of Mn2 ions (red). The tyrosine residues to
which adenyl groups can be attached are colored red. (a) Top view of
molecule. (b) Side view showing the six nearest subunits.
8.9
lated enzyme, glutamine synthetase is not very active. Removal
of AMP groups produces more active deadenylylated glutamine
synthetase, and glutamine is formed. The glutamine synthetase
system differs from the phosphorylase system in two ways:
(1) AMP is used as the modifying agent, and (2) the modified
form of glutamine synthetase is less active. Glutamine synthetase
also is allosterically regulated. Glutamine synthetase and its role in nitrogen metabolism (pp. 211–12)
End product P
F
G
–
–
E
B
–
The rate of many metabolic pathways is adjusted through control
of the activity of the regulatory enzymes described in the preceding section. Every pathway has at least one pacemaker enzyme
that catalyzes the slowest or rate-limiting reaction in the pathway.
Because other reactions proceed more rapidly than the pacemaker
reaction, changes in the activity of this enzyme directly alter the
speed with which a pathway operates. Usually the first step in a
pathway is a pacemaker reaction catalyzed by a regulatory enzyme. The end product of the pathway often inhibits this regulatory enzyme, a process known as feedback inhibition or end
product inhibition. Feedback inhibition ensures balanced production of a pathway end product. If the end product becomes too
concentrated, it inhibits the regulatory enzyme and slows its own
synthesis. As the end product concentration decreases, pathway
activity again increases and more product is formed. In this way
feedback inhibition automatically matches end product supply
with the demand. The previously discussed E. coli aspartate carbamoyltransferase is an excellent example of end product or feedback inhibition.
Frequently a biosynthetic pathway branches to form more
than one end product. In such a situation the synthesis of pathway end products must be coordinated precisely. It would not do
to have one end product present in excess while another is lacking. Branching biosynthetic pathways usually achieve a balance
between end products through the use of regulatory enzymes at
branch points (figure 8.27). If an end product is present in excess, it often inhibits the branch-point enzyme on the sequence
leading to its formation, in this way regulating its own formation
without affecting the synthesis of other products. In figure 8.27
notice that both products also inhibit the initial enzyme in the
pathway. An excess of one product slows the flow of carbon into
the whole pathway while inhibiting the appropriate branch-point
enzyme. Because less carbon is required when a branch is not
functioning, feedback inhibition of the initial pacemaker enzyme
169
End product Q
There are some advantages to using covalent modification
for the regulation of enzyme activity. These interconvertible enzymes often are also allosteric. Because each form can respond
differently to allosteric effectors, systems of covalently modified
enzymes are able to respond to more stimuli in varied and sophisticated ways. Regulation can also be exerted on the enzymes
that catalyze the covalent modifications, which adds a second
level of regulation to the system.
Feedback Inhibition
Control of Enzyme Activity
–
Substrate A
Figure 8.27 Feedback Inhibition. Feedback inhibition in a
branching pathway with two end products. The branch-point
enzymes, those catalyzing the conversion of intermediate E to F and
G, are regulated by feedback inhibition. Products P and Q also inhibit
the initial reaction in the pathway. A colored line with a minus sign at
one end indicates that an end product, P or Q, is inhibiting the
enzyme catalyzing the step next to the minus. See text for further
explanation.
helps match the supply with the demand in branching pathways.
The regulation of multiple branched pathways is often made
even more sophisticated by the presence of isoenzymes, different enzymes that catalyze the same reaction. The initial pacemaker step may be catalyzed by several isoenzymes, each under
separate and independent control. In such a situation an excess
of a single end product reduces pathway activity but does not
completely block pathway function because some isoenzymes
are still active.
1. Define the following: allosteric enzyme, effector or modulator,
and [S]0.5 or K0.5.
2. How can regulatory enzymes be influenced by reversible covalent
modification? What groups are used for this purpose with
glycogen phosphorylase and glutamine synthetase, and which
forms of these enzymes are active?
3. What is a pacemaker enzyme? Feedback inhibition? How does
feedback inhibition automatically adjust the concentration of a
pathway end product? What are isoenzymes and why are they
important in pathway regulation?
170
Chapter 8
Metabolism: Energy, Enzymes, and Regulation
Summary
1. Energy is the capacity to do work. Living cells
carry out three major kinds of work: chemical
work of biosynthesis, transport work, and
mechanical work.
2. The ultimate source of energy for most
microbes is sunlight trapped by autotrophs and
used to form organic material from CO2.
Photoautotrophs are then consumed by
chemoheterotrophs.
3. ATP is the major energy currency and
connects energy-generating processes with
energy-using processes (figure 8.3).
4. The first law of thermodynamics states that
energy is neither created nor destroyed.
5. The second law of thermodynamics states that
changes occur in such a way that the
randomness or disorder of the universe increases
to the maximum possible. That is, entropy
always increases during spontaneous processes.
6. The first and second laws can be combined to
determine the amount of energy made
available for useful work.
G H T· S
In this equation the change in free energy
(G) is the energy made available for useful
work, the change in enthalpy (H) is the
change in heat content, and the change in
entropy is S.
7. The standard free energy change (Go′) for a
chemical reaction is directly related to the
equilibrium constant.
8. In exergonic reactions Go′ is negative and
the equilibrium constant is greater than one;
the reaction goes to completion as written.
Endergonic reactions have a positive Go′
and an equilibrium constant less than one
(figure 8.5).
9. In oxidation-reduction (redox) reactions,
electrons move from a donor, the reducing
agent or reductant, to an acceptor, the
oxidizing agent or oxidant. The standard
reduction potential measures the tendency of
the reducing agent to give up electrons.
10. Redox couples with more negative reduction
potentials donate electrons to those with more
positive potentials, and energy is made
available during the transfer (figure 8.7).
11. Some most important electron carriers in cells
are NAD, NADP, FAD, FMN, coenzyme Q,
cytochromes, and the nonheme iron proteins.
12. Enzymes are protein catalysts that catalyze
specific reactions.
13. Enzymes consist of a protein component, the
apoenzyme, and often a nonprotein cofactor
that may be a prosthetic group, a coenzyme, or
a metal activator.
14. Enzymes speed reactions by binding
substrates at their active sites and lowering the
activation energy (figure 8.14).
15. The rate of an enzyme-catalyzed reaction
increases with substrate concentration at low
substrate levels and reaches a plateau (the
maximum velocity) at saturating substrate
concentrations. The Michaelis constant is the
16.
17.
18.
19.
20.
21.
22.
23.
substrate concentration that the enzyme requires
to achieve half maximal velocity (figure 8.17).
Enzymes have pH and temperature optima for
activity.
Enzyme activity can be slowed by competitive
and noncompetitive inhibitors.
The regulation of metabolism keeps cell
components in proper balance and conserves
metabolic energy and material.
The localization of metabolites and enzymes
in different parts of the cell, called metabolic
channeling, influences pathway activity. A
common channeling mechanism is
compartmentation.
Regulatory enzymes are usually allosteric
enzymes, enzymes in which an effector or
modulator binds reversibly to a regulatory site
separate from the catalytic site and causes a
conformational change in the enzyme to alter
its activity (figure 8.21).
Aspartate carbamoyltransferase is an allosteric
enzyme that is inhibited by CTP and activated
by ATP.
Enzyme activity also can be regulated by
reversible covalent modification. Two
examples of such regulation are glycogen
phosphorylase (phosphate addition) and
glutamine synthetase (AMP addition).
The first enzyme in a pathway and enzymes at
branch points often are subject to feedback
inhibition by one or more end products.
Excess end product slows its own synthesis
(figure 8.27).
Key Terms
activation energy 162
active site 162
adenosine diphosphate (ADP) 155
adenosine 5′-triphosphate (ATP) 155
aerobic respiration 154
allosteric enzymes 165
apoenzyme 161
calorie 155
catalyst 161
catalytic site 162
chemical work 154
coenzyme 161
coenzyme Q or CoQ (ubiquinone) 159
cofactor 161
compartmentation 165
competitive inhibitor 164
cytochrome 159
denaturation 163
effector or modulator 165
end product inhibition 169
endergonic reaction 156
energy 154
enthalpy 156
entropy 156
enzyme 161
enzyme-substrate complex 162
equilibrium 156
equilibrium constant (Keq) 156
exergonic reaction 156
feedback inhibition 169
ferredoxin 159
first law of thermodynamics 155
flavin adenine dinucleotide (FAD) 159
flavin mononucleotide (FMN) 159
free energy change 156
high-energy molecule 157
holoenzyme 161
isoenzymes 169
joule 155
mechanical work 154
metabolic channeling 165
Michaelis constant (Km) 163
nicotinamide adenine dinucleotide (NAD) 157
nicotinamide adenine dinucleotide phosphate
(NADP) 158
noncompetitive inhibitor 164
nonheme iron protein 159
oxidation-reduction (redox) reaction 157
oxidizing agent (oxidant) 157
pacemaker enzyme 169
phosphate group transfer potential 157
photosynthesis 154
product 161
prosthetic group 161
reducing agent (reductant) 157
regulatory site 165
reversible covalent modification 167
second law of thermodynamics 156
standard free energy change 156
standard reduction potential 157
substrate 161
thermodynamics 155
transition-state complex 162
transport work 154
Additional Reading
Questions for Thought and Review
1. Describe in general terms how energy from
sunlight is spread throughout the biosphere.
2. What sources of energy, other than sunlight,
are used by microorganisms?
3. Under what conditions would it be possible to
create more order in a system without
violating the second law of thermodynamics?
4. Do living cells increase randomness or entropy
within themselves? In the environment?
5. Suppose that a chemical reaction had a large
negative Go′ value. What would this indicate
about its equilibrium constant? If displaced
from equilibrium, would it proceed rapidly to
completion? Would much or little free energy
be made available?
6. Will electrons ordinarily move in an electron
transport chain from cytochrome c
(E′0 210 mV) to O2 (E′0 820 mV)
or in the opposite direction?
7. If a person had a niacin deficiency, what
metabolic process might well be adversely
affected? Why?
8. Draw a diagram showing how enzymes
catalyze reactions by altering the activation
energy. What is a transition-state complex?
Use the diagram to discuss why enzymes do
not change the equilibria of the reactions they
catalyze.
9. What special properties might an enzyme
isolated from a psychrophilic bacterium have?
Will enzymes need to lower the activation
energy more or less in thermophiles than in
psychrophiles?
10. How might a substrate be able to regulate the
activity of the enzyme using it?
11. Describe how E. coli aspartate
carbamoyltransferase is regulated, both in
terms of the effects of modulators and the
mechanism by which they exert their
influence.
12. What is the significance of the fact that
regulatory enzymes often are located at
pathway branch points?
171
Critical Thinking Questions
1. How could electron transport be driven in the
opposite direction? Why would it be desirable
to do this?
2. Take a look at the structures of
macromolecules (appendix I). Which type has
the most electrons to donate? Why are
carbohydrates usually the primary source of
electrons for nonautotrophic bacteria?
3. Most enzymes do not operate at their
biochemical optima inside cells. Why not?
Additional Reading
General
Becker, W. M.; Kleinsmith, L.; and Hardin, J. 2000.
The world of the cell, 4th ed. Redwood City,
Calif.: Benjamin Cummings.
Garrett, R. H., and Grisham, C. H. 1999.
Biochemistry 2d ed. New York: Saunders.
Lehninger, A. L.; Nelson, D. L.; and Cox, M. M.
1993. Principles of biochemistry, 2d ed. New
York: Worth Publishers.
Lodish, H.; Baltimore, D.; Berk, A.; Zipursky,
S. L.; Matsudaira, P.; and Darnell, J. 1999.
Molecular cell biology, 4th ed. New York:
Scientific American Books.
Mathews, C. K., and van Holde, K. E. 1996.
Biochemistry, 2d ed. Menlo Park, Calif.:
Benjamin/Cummings.
Moran, L. A.; Scrimgeour, K. G.; Horton, H. R.;
Ochs, R. S.; and Rawn, J. D. 1994.
Biochemistry. Englewood Cliffs, N.J.: Neil
Patterson Publishers/Prentice-Hall.
Neidhardt, F. C.; Ingraham, J. L.; and Schaechter, M.
1990. Physiology of the bacterial cell: A
molecular approach. Sunderland, Mass.:
Sinauer Associates.
Stryer, L. 1995. Biochemistry, 4th ed. New York:
Freeman.
Voet, D., and Voet, J. G. 1995. Biochemistry, 2d ed.
New York: John Wiley and Sons.
Zubay, G. 1998. Biochemistry, 4th ed. Dubuque,
Iowa: WCB/McGraw-Hill.
8.6
Enzymes
Boyer, Paul D., editor. 1970–1987. The enzymes.
San Diego: Academic Press.
Branden, C., and Tooze, J. 1991. Introduction to
protein structure. New York: Garland Publishing.
Fersht, A. 1984. Enzyme structure and mechanism,
2d ed. San Francisco: W. H. Freeman.
International Union of Biochemistry and Molecular
Biology. 1992. Enzyme nomenclature. San
Diego: Academic Press.
Kraut, J. 1988. How do enzymes work? Science
242:533–39.
Neidleman, S. L. 1989. Enzymes under stress. ASM
News 55(2):67–70.
Walsh, C. 1979. Enzymatic reaction mechanisms.
San Francisco: W. H. Freeman.
8.9
Control of Enzyme Activity
Kantrowitz, E. R., and Lipscomb, W. N. 1988.
Escherichia coli aspartate transcarbamylase:
The relation between structure and function.
Science 241:669–74.
Koshland, D. E., Jr. 1973. Protein shape and
biological control. Sci. Am. 229(4):52–64.
Saier, M. H., Jr.; Wu, L.-F.; and Reizer, J. 1990.
Regulation of bacterial physiological
processes by three types of protein
phosphorylating systems. Trends Biochem.
Sci. 15:391–95.